Gaps in our understanding of how vagal afferents to the small intestinal mucosa detect luminal stimuli.

Vagal afferents to the gastrointestinal tract are crucial for the regulation of food intake, signaling negative feedback that contributes to satiation and positive feedback that produces appetition and reward. Vagal afferents to the small intestinal mucosa contribute to this regulation by sensing luminal stimuli and reporting this information to the brain. These afferents respond to mechanical, chemical, thermal, pH, and osmolar stimuli, as well as to bacterial products and immunogens. Surprisingly, little is known about how these stimuli are transduced by vagal mucosal afferents or how their transduction is organized among these afferents' terminals. Furthermore, the effects of stimulus concentration ranges or physiological stimuli on vagal activity have not been examined for some of these stimuli. Also, detection of luminal stimuli has rarely been examined in rodents, which are most frequently used for studying small intestinal innervation. Here we review what is known about stimulus detection by vagal mucosal afferents and illustrate the complexity of this detection using nutrients as an exemplar. The accepted model proposes that nutrients bind to taste receptors on enteroendocrine cells (EECs), which excite them, causing the release of hormones that stimulate vagal mucosal afferents. However, evidence reviewed here suggests that although this model accounts for many aspects of vagal signaling about nutrients, it cannot account for all aspects. A major goal of this review is therefore to evaluate what is known about nutrient absorption and detection and, based on this evaluation, identify candidate mucosal cells and structures that could cooperate with EECs and vagal mucosal afferents in stimulus detection.

Aberrant enteric neuromuscular system and dysbiosis in amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis is a neuromuscular disease characterized by the progressive death of motor neurons and muscle atrophy. The gastrointestinal symptoms in ALS patients were largely ignored or underestimated. The relationship between the enteric neuromuscular system and microbiome in ALS progression is unknown. We performed longitudinal studies on the enteric neuron system (ENS) and microbiome in the ALS human-SOD1G93A (Superoxide Dismutase 1) transgenic mice. We treated age-matched wild-type and ALS mice with butyrate or antibiotics to investigate the microbiome and neuromuscular functions. We examined intestinal mobility, microbiome, an ENS marker GFAP (Glial Fibrillary Acidic Protein), a smooth muscle marker (SMMHC, Smooth Muscle Myosin Heavy Chain), and human colonoids. The distribution of human-G93A-SOD1 protein was tested as an indicator of ALS progression. At 2-month-old before ALS onset, SOD1G93A mice had significantly lower intestinal mobility, decreased grip strength, and reduced time in the rotarod. We observed increased GFAP and decreased SMMHC expression. These changes correlated with consistent increased aggregation of mutated SOD1G93A in the colon, small intestine, and spinal cord. Butyrate or antibiotics treated SOD1G93A mice had a significantly longer latency to fall in the rotarod test, reduced SOD1G93A aggregation, and enhanced enteric neuromuscular function. Feces from 2-month-old SOD1G93A mice significantly enhanced SOD1G93A aggregation in human colonoids transfected with a SOD1G93A-GFP plasmid. Longitudinal studies of microbiome data further showed the altered bacterial community related to autoimmunity (e.g., Clostridium sp. ASF502, Lachnospiraceae bacterium A4), inflammation (e.g., Enterohabdus Muris,), and metabolism (e.g., Desulfovibrio fairfieldensis) at 1- and 2-month-old SOD1G93A mice, suggesting the early microbial contribution to the pathological changes. We have demonstrated a novel link between the microbiome, hSOD1G93A aggregation, and intestinal mobility. Dysbiosis occurred at the early stage of the ALS mice before observed mutated-SOD1 aggregation and dysfunction of ENS. Manipulating the microbiome improves the muscle performance of SOD1G93A mice. We provide insights into the fundamentals of intestinal neuromuscular function and microbiome in ALS.

Mouse Enteric Neuronal Cell Culture.

In the enteric nervous system, there exist a huge number of local intrinsic neurons, which control the gastrointestinal functions. Culture of enteric neurons provides a good model system for physiological, electrophysiological, and pharmacological studies. Here, we describe two methods to obtain sufficient enteric neurons from mouse myenteric plexuses by directly culturing primary neurons or inducing neuronal differentiation of enteric neural stem/progenitor cells.

Small intestine remodeling in male Goto-Kakizaki rats.

BACKGROUND: Obesity is associated with the development of insulin resistance (IR) and type-2 diabetes mellitus (T2DM); however, not all patients with T2DM are obese. The Goto-Kakizaki (GK) rat is an experimental model of spontaneous and non-obese T2DM. There is evidence that the intestine contributes to IR development in GK animals. This information prompted us to investigate small intestine remodeling in this animal model. METHODS: Four-month-old male Wistar (control) and GK rats were utilized for the present study. After removing the small intestine, the duodenum, proximal jejunum, and distal ileum were separated. We then measured villi and muscular and mucosa layer histomorphometry, goblet cells abundance, total myenteric and submucosal neuron populations, and inflammatory marker expression in the small intestinal segments and intestinal transit of both groups of animals. KEY RESULTS: We found that the GK rats exhibited decreased intestinal area (p < 0.0001), decreased crypt depth in the duodenum (p = 0.01) and ileum (p < 0.0001), increased crypt depth in the jejunum (p < 0.0001), longer villi in the jejunum and ileum (p < 0.0001), thicker villi in the duodenum (p < 0.01) and ileum (p < 0.0001), thicker muscular layers in the duodenum, jejunum, and ileum (p < 0.0001), increased IL-1ß concentrations in the duodenum and jejunum (p < 0.05), and increased concentrations of NF-κB p65 in the duodenum (p < 0.01), jejunum and ileum (p < 0.05). We observed high IL-1ß reactivity in the muscle layer, myenteric neurons, and glial cells of the experimental group. GK rats also exhibited a significant reduction in submucosal neuron density in the jejunum and ileum, ganglionic hypertrophy in all intestinal segments studied (p < 0.0001), and a slower intestinal transit (about 25%) compared to controls. CONCLUSIONS: The development of IR and T2DM in GK rats is associated with small intestine remodeling that includes marked alterations in small intestine morphology, local inflammation, and reduced intestinal transit.

Genetic background-dependent abnormalities of the enteric nervous system and intestinal function in Kif26a-deficient mice.

The Kif26a protein-coding gene has been identified as a negative regulator of the GDNF-Ret signaling pathway in enteric neurons. The aim of this study was to investigate the influence of genetic background on the phenotype of Kif26a-deficient (KO, -/-) mice. KO mice with both C57BL/6 and BALB/c genetic backgrounds were established. Survival rates and megacolon development were compared between these two strains of KO mice. Functional bowel assessments and enteric neuron histopathology were performed in the deficient mice. KO mice with the BALB/c genetic background survived more than 400 days without evidence of megacolon, while all C57BL/6 KO mice developed megacolon and died within 30 days. Local enteric neuron hyperplasia in the colon and functional bowel abnormalities were observed in BALB/c KO mice. These results indicated that megacolon and enteric neuron hyperplasia in KO mice are influenced by the genetic background. BALB/c KO mice may represent a viable model for functional gastrointestinal diseases such as chronic constipation, facilitating studies on the underlying mechanisms and providing a foundation for the development of treatments.

Liraglutide accelerates colonic transit in people with type 1 diabetes and polyneuropathy: A randomised, double-blind, placebo-controlled trial.

BACKGROUND: Glucagon-like peptide-1 receptor agonists, such as liraglutide, reduce hyperglycaemia and induce weight loss and are used as a treatment in diabetes. However, common adverse effects include nausea, loss of appetite and prolonged gastric emptying. It is not known whether these changes are centrally generated or if liraglutide alters the enteric motility. OBJECTIVE: To investigate the effects of liraglutide on gastrointestinal function and symptoms. METHODS: A total of 48 adults with type 1 diabetes and confirmed distal symmetric polyneuropathy were randomised to receive liraglutide 1.8 mg/day or placebo for 26 weeks. Regional transit times and motility indexes were assessed with a wireless motility capsule, whereas symptoms were evaluated using the validated gastroparesis cardinal symptom index. RESULTS: Liraglutide treatment reduced large bowel transit time (31.7%, p = 0.04) and decreased motility index (6.1%, p = 0.04) compared to placebo, whereas the groups did not differ in gastric emptying or small-bowel transit times. Liraglutide increased postprandial fullness with 29% (p = 0.01). Increased small bowel transit time was associated with decreased bloating (p = 0.008). CONCLUSION: Liraglutide accelerates large bowel transit and decreases motility index, which may indicate better coordination of propulsive motility. This potentially improves the function of the enteric nervous system, leading to normalised colonic function and positive effects in type 1 diabetes.

Sensory Nociceptive Neurons Contribute to Host Protection During Enteric Infection With Citrobacter rodentium.

BACKGROUND: Neurons are an integral component of the immune system that functions to coordinate responses to bacterial pathogens. Sensory nociceptive neurons that can detect bacterial pathogens are found throughout the body with dense innervation of the intestinal tract. METHODS: In this study, we assessed the role of these nerves in the coordination of host defenses to Citrobacter rodentium. Selective ablation of nociceptive neurons significantly increased bacterial burden 10 days postinfection and delayed pathogen clearance. RESULTS: Because the sensory neuropeptide CGRP (calcitonin gene-related peptide) regulates host responses during infection of the skin, lung, and small intestine, we assessed the role of CGRP receptor signaling during C rodentium infection. Although CGRP receptor blockade reduced certain proinflammatory gene expression, bacterial burden and Il-22 expression was unaffected. CONCLUSIONS: Our data highlight that sensory nociceptive neurons exert a significant host protective role during C rodentium infection, independent of CGRP receptor signaling.

Neural motor complexes propagate continuously along the full length of mouse small intestine and colon.

Cyclical propagating waves of muscle contraction have been recorded in isolated small intestine or colon, referred to here as motor complexes (MCs). Small intestinal and colonic MCs are neurogenic, occur at similar frequencies, and propagate orally or aborally. Whether they can be coordinated between the different gut regions is unclear. Motor behavior of whole length mouse intestines, from duodenum to terminal rectum, was recorded by intraluminal multisensor catheter. Small intestinal MCs were recorded in 27/30 preparations, and colonic MCs were recorded in all preparations (n = 30) with similar frequencies (0.54 ± 0.03 and 0.58 ± 0.02 counts/min, respectively). MCs propagated across the ileo-colonic junction in 10/30 preparations, forming "full intestine" MCs. The cholinesterase inhibitor physostigmine increased the probability of a full intestine MC but had no significant effect on frequency, speed, or direction. Nitric oxide synthesis blockade by Nω-nitro-l-arginine, after physostigmine, increased MC frequency in small intestine only. Hyoscine-resistant MCs were recorded in the colon but not small intestine (n = 5). All MCs were abolished by hexamethonium (n = 18) or tetrodotoxin (n = 2). The enteric neural mechanism required for motor complexes is present along the full length of both the small and large intestine. In some cases, colonic MCs can be initiated in the distal colon and propagate through the ileo-colonic junction, all the way to duodenum. In conclusion, the ileo-colonic junction provides functional neural continuity for propagating motor activity that originates in the small or large intestine.NEW & NOTEWORTHY Intraluminal manometric recordings revealed motor complexes can propagate antegradely or retrogradely across the ileo-colonic junction, spanning the entire small and large intestines. The fundamental enteric neural mechanism(s) underlying cyclic motor complexes exists throughout the length of the small and large intestine.

Development of the intrinsic innervation of the small bowel mucosa and villi.

Detection of nutritional and noxious food components in the gut is a crucial component of gastrointestinal function. Contents in the gut lumen interact with enteroendocrine cells dispersed throughout the gut epithelium. Enteroendocrine cells release many different hormones, neuropeptides, and neurotransmitters that communicate either directly or indirectly with the central nervous system and the enteric nervous system, a network of neurons and glia located within the gut wall. Several populations of enteric neurons extend processes that innervate the gastrointestinal lamina propria; however, how these processes develop and begin to transmit information from the mucosa is not fully understood. In this study, we found that Tuj1-immunoreactive neurites begin to project out of the myenteric plexus at embryonic day (E)13.5 in the mouse small intestine, even before the formation of villi. Using live calcium imaging, we discovered that neurites were capable of transmitting electrical information from stimulated villi to the plexus by E15.5. In unpeeled gut preparations where all layers were left intact, we also mimicked the basolateral release of 5-HT from enteroendocrine cells, which triggered responses in myenteric cell bodies at postnatal day (P)0. Altogether, our results show that enteric neurons extend neurites out of the myenteric plexus early during mouse enteric nervous system development, innervating the gastrointestinal mucosa, even before villus formation in mice of either sex. Neurites are already able to conduct electrical information at E15.5, and responses to 5-HT develop postnatally.NEW & NOTEWORTHY How enteric neurons project into the gut mucosa and begin to communicate with the epithelium during development is not known. Our study shows that enteric neurites project into the lamina propria as early as E13.5 in the mouse, before development of the submucous plexus and before formation of intestinal villi. These neurites are capable of transmitting electrical signals back to their cell bodies by E15.5 and respond to serotonin applied to neurite terminals by birth.

Abdominal vagotomy reveals majority of small intestinal mucosal afferents labeled in nav 1.8cre-rosa26tdTomato mice are vagal in origin.

Vagal afferents innervating the small intestinal mucosa regulate feeding, gastrointestinal (GI) digestive, and immune functions. Their anatomical-functional characterization has been impeded by the inability to selectively label and manipulate them. Nav 1.8-Cre-tdTomato mice label 80% of nodose and dorsal root ganglia neurons. Here, the origin of these neuron's terminals and their distribution in the small intestinal mucosa were examined by quantitatively comparing tdTomato-labeled innervation in nonoperated (control), subdiaphragmatic vagotomy (VAGX), and sham-operated mice. Control mice exhibited a large proximal-to-distal decrease and a moderate mesentery-to-antimesentery decrease in villus innervation. VAGX reduced this innervation to a greater degree proximally (91-93%) than distally (65-72%), resulting in flat proximal-distal distributions. Therefore, estimates of vagal villus afferent distributions (control minus VAGX) paralleled control distributions, but were slightly reduced in magnitude. Compared with villus afferents, crypt innervation exhibited a muted proximal-to-distal decrease in control mice and a smaller loss after VAGX (45-48%). Sham-operated mice exhibited similar distributions of villus and crypt afferents as control mice, suggesting surgery did not contribute to the effects of VAGX. Most crypt and villus afferent terminals along the entire proximal-distal small intestinal axis had similar morphology to those previously reported in the proximal duodenum, but the density of terminal branches varied. Our findings suggest the majority of small intestinal mucosal innervation labeled in Nav 1.8-Cre-tdTomato mice is vagal in origin. Therefore, these mice will be valuable for studying vagal mucosal afferent morphology, interactions with other GI elements, plasticity, and function.

Shrinkage of the myenteric neurons of the small intestine in patients with multiple system atrophy.

This study aimed to determine whether enteric neurons are involved in multiple system atrophy (MSA). Four-µm-thick slices of small intestine were prepared from 10%-formalin-fixed and paraffin-embedded materials obtained from autopsied cases. Enteric neurons were stained using an anti-peripherin antibody. Immunostaining of phosphorylated α-synuclein was also performed. Areas of the cytoplasm and nucleus that showed nucleoli were measured using computer software. Both areas of myenteric neurons were significantly smaller in MSA cases (n = 3) than in control subjects (n = 3) (P < 0.0001); however, no deposits of phosphorylated α-synuclein were observed. These findings suggest that myenteric neurons in MSA are affected independent of α-synuclein accumulation.

Refeeding reverses fasting-induced remodeling of afferent nerve activity in rat small intestine.

Intestinal afferents play an important role in coordinating intestinal motor control. Fasting induces morpho-mechanical intestinal remodeling. This study aimed to characterize the effect of fasting and refeeding on mechanosensitivity in mesenteric afferent nerves in isolated Sprague-Dawley rat jejunum. A control group fed ad libitum, a group fasted for 7 days and a group refed 7 days after 7 days fasting were studied. Jejunal segments were used for electrophysiological, histomorphological and mechanical studies. Mesenteric afferent nerve firing was recorded during a ramp distension up to 40 mmHg luminal pressure. Multiunit afferent recordings were separated into low threshold and wide-dynamic-range single-unit activity. Intestinal deformation (strain), bowel distension load (stress) and firing frequency of mesenteric afferent nerve bundles [spike rate increase ratio (SRIR)] were compared among groups. Fasting induced intestinal histomorphometric remodeling, which was reversed by refeeding. The firing frequency increased with distension in all groups. SRIR was largest in the fasting group (P < 0.05). Compared to the control group, fasting increased afferent activity in whole nerve bundles and wide-dynamic-range units at high degrees of distensions (P < 0.05 at pressure 40 mmHg; P < 0.05 at strain 1.2; P < 0.01 at stress 8 kPa). Refeeding reversed the fasting-induced afferent hypersensitivity and the shift between receptor subtypes. In conclusion, refeeding reversed fasting-induced remodeling.

Delayed appearance of mature ganglia in an infant with an atypical presentation of total colonic and small bowel aganglionosis: a case report.

BACKGROUND: Total colonic and small bowel aganglionosis (TCSA) occurs in less than 1% of all Hirschsprung's disease patients. Currently, the mainstay of treatment is surgery. However, in patients with TCSA, functional outcomes are often poor. A characteristic transition zone in TCSA can be difficult to identify which may complicate surgery and may often require multiple operations. CASE PRESENTATION: We present the case of a male infant who was diagnosed with biopsy-proven total colonic aganglionosis with extensive small bowel involvement as a neonate. The patient was diverted at one month of age based on leveling biopsies at 10 cm from the Ligament of Treitz. At 7 months of age, during stoma revision for a prolapsed stoma, intra-operative peristalsis was observed in nearly the entire length of the previously aganglionic bowel, and subsequent biopsies demonstrated the appearance of mature ganglion cells in a previously aganglionic segment. CONCLUSIONS: TCSA remains a major challenge for pediatric surgeons. Our case introduces new controversy to our understanding of aganglionosis. Our observations warrant further research into the possibility of post-natal ganglion maturation and encourage surgeons to consider a more conservative surgical approach.

Method for Detecting Hyaluronan in Isolated Myenteric Plexus Ganglia of Adult Rat Small Intestine.

The cellular components of the enteric nervous system (ENS), namely enteric neurons and glia, display plasticity and respond to environmental cues deriving from growth factors, extracellular matrix (ECM) molecules, and cell-surface molecules, both in physiological and pathological conditions. ECM, in particular, provides an important framework for the enteric microenvironment and influences the homeostasis of myenteric neuronal circuitries. Isolation of pure myenteric plexus preparations from adult tissue permits to investigate changes in the ENS involving specific ECM, such as hyaluronan. This approach is based upon the possibility to isolate myenteric ganglia from the intestinal wall of either adult animals or humans, after microdissection and subsequent enzymatic digestion of the tissue. Enteric ganglia are free of connective tissue, extracellular collagen, and blood vessels, and thus treatment of intact intestinal segments with highly purified collagenases permits ganglia isolation from the surrounding smooth muscle cells. In this chapter, we describe methods for visualizing HA in isolated primary cultures of adult rat small intestine myenteric ganglia.

Bowel Motility After Injury to the Superior Mesenteric Plexus During D3 Extended Mesenterectomy.

BACKGROUND: Improvement of lymphadenectomy in right colectomy requires removal of all tissue surrounding the superior mesenteric vessels beneath the pancreatic notch. Short- and long-term bowel motility disorders after D3 extended mesenterectomy with consecutive superior mesenteric plexus transection are studied. METHODS: Patients without pre-existing motility disorders undergoing D3 extended mesenterectomy were examined 3 times using the wireless motility capsule: before, at 3 wk, and 6 mo after surgery. Segmental transit times and contractility were analyzed using mixed effect modeling. Correlation between contractility and transit time was assessed by the Pearson correlation coefficient. RESULTS: Fifteen patients (4 men), with median age 62 y, were included. Mean values for the three consecutive examinations are as follows. Gastric transit time increased from 237 to 402 and 403 min, respectively. Small bowel transit time decreased from 246 to 158 (P < 0.01) and 199 (P = 0.03) min, respectively. Colonic transit time decreased from 1742 to 1450 and 1110 (P = 0.02) min, respectively. Gastric contractions per minute (CPM) varied from 1.73 to 1.05 (P = 0.01) and 2.47 (P < 0.01), respectively. Small bowel CPM decreased from 3.43 to 2.68 and 3.34, respectively. Colonic CPM ranged from 1.59 to 1.45 and 1.91 (P = 0.08), respectively. Correlation between small bowel (SB) transit time and CPM was -0.45 (P = 0.09) preoperatively, and -0.03 (P = 0.91) 6 mo postoperatively. CONCLUSIONS: Extrinsic SB denervation leads to significantly accelerated SB transit, reduced contractility, and disturbed correlation between transit time and contractility early after denervation. Both number of contractions and transit time in the denervated SB show a clear tendency toward normalization at 6 mo.

The Effect of Gastric Electrical Stimulation on Small Bowel Motility in Patients With Gastroparesis and Concomitant Pancreatic and Small Bowel Dysfunction: From Animal Model to Human Application.

BACKGROUND/AIMS: Patients with gastroparesis often have biliary/pancreatic and small bowel symptoms but the effects of gastric electrical stimulation on small bowel electrical activity of the mid-gut have not been studied. Animal model aim: Establish gastric and upper small bowel/biliary slow wave activity relationships with electrical stimulation. Human study aim: Demonstrate improvement in symptoms associated with proximal small bowel dysmotility in gastric stimulated patients. MATERIALS AND METHODS: Animal model: In vivo evoked responses of duodenal and Sphincter of Oddi measures recorded during gastric electrical stimulation in a nonsurvival swine model (N = 3). High-resolution electrical slow wave mapping of frequency, amplitude, and their ratio, for duodenal and Sphincter of Oddi electrical activity were recorded. Human study: Patients (N = 8) underwent temporary gastric stimulation with small bowel electrodes. Subjective and objective data was collected before and after temporary gastric stimulation. Symptom scores, gastric emptying times, and mucosal electrograms via low-resolution mapping were recorded. RESULTS: Animal gastric stimulation resulted in some changes in electrical activity parameters, especially with the highest energies delivered but the changes were not statistically significant. Human study revealed improvement in symptom and illness severity scores, and changes in small bowel mucosal slow wave activity. CONCLUSIONS: Gastric electrical stimulation in an animal model seems to show nonsignificant effects small bowel slow wave activity and myoelectric signaling, suggesting the existence of intrinsic neural connections. Human data shows more significance, with possible potential for therapeutic use of electrical stimulation in patients with gastroparesis and pancreato-biliary and small bowel symptoms of the mid-gut. This study was limited by the nonsurvival pig model, small sample size, and open label human study.

The enteric neural crest progressively loses capacity to form enteric nervous system.

Cells of the vagal neural crest (NC) form most of the enteric nervous system (ENS) by a colonising wave in the embryonic gut, with high cell proliferation and differentiation. Enteric neuropathies have an ENS deficit and cell replacement has been suggested as therapy. This would be performed post-natally, which raises the question of whether the ENS cell population retains its initial ENS-forming potential with age. We tested this on the avian model in organ culture in vitro (3 days) using recipient aneural chick midgut/hindgut combined with ENS-donor quail midgut or hindgut of ages QE5 to QE10. ENS cells from young donor tissues (≤ QE6) avidly colonised the aneural recipient, but this capacity dropped rapidly 2-3 days after the transit of the ENS cell wavefront. This loss in capability was autonomous to the ENS population since a similar decline was observed in ENS cells isolated by HNK1 FACS. Using QE5, 6, 8 and 10 midgut donors and extending the time of assay to 8 days in chorio-allantoic membrane grafts did not produce 'catch up' colonisation. NC-derived cells were counted in dissociated quail embryo gut and in transverse sections of chick embryo gut using NC, neuron and glial marker antibodies. This showed that the decline in ENS-forming ability correlated with a decrease in proportion of ENS cells lacking both neuronal and glial differentiation markers, but there were still large numbers of such cells even at stages with low colonisation ability. Moreover, ENS cells in small numbers from young donors were far superior in colonisation ability to larger numbers of apparently undifferentiated cells from older donors. This suggests that the decline of ENS-forming ability has both quantitative and qualitative aspects. In this case, ENS cells for cell therapies should aim to replicate the embryonic ENS stage rather than using post-natal ENS stem/progenitor cells.

Rat enteric glial cells express novel isoforms of Interleukine-7 regulated during inflammation.

BACKGROUND: Neuroimmune interactions are essential to maintain gut homeostasis and prevent intestinal disorders but so far, the impact of enteric glial cells (EGC) on immune cells remains a relatively unexplored area of research. As a dysregulation of critical cytokines such as interleukine-7 (IL-7) was suggested to exacerbate gut chronic inflammation, we investigated whether EGC could be a source of IL-7 in the gastrointestinal tract. METHODS: Expression of IL-7 in the rat enteric nervous system was analyzed by immunochemistry and Q-PCR. IL-7 variants were cloned and specific antibodies against rat IL-7 isoforms were raised to characterize their expression in the submucosal plexus. IL-7 isoforms were produced in vitro to analyze their impact on T-cell survival. KEY RESULTS: Neurons and glial cells of the rat enteric nervous system expressed IL-7 at both mRNA and protein levels. Novel rat IL-7 isoforms with distinct C-terminal parts were detected. Three of these isoforms were found in EGC or in both enteric neurons and EGC. Exposure of EGC to pro-inflammatory cytokines (IL-1ß and/or TNFα) induced an upregulation of all IL-7 isoforms. Interestingly, time-course and intensity of the upregulation varied according to the presence or absence of exon 5a in IL-7 variants. Functional analysis on T lymphocytes revealed that only canonical IL-7 protects T cells from cell death. CONCLUSIONS AND INFERENCES: IL-7 and its variants are expressed by neurons and glial cells in the enteric nervous system. Their distinct expression and upregulation in inflammatory conditions suggest a role in gut homeostasis which could be critical in case of chronic inflammatory diseases.

Preoperative administration of the 5-HT4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons.

OBJECTIVES: Vagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation. Here, we evaluated if 5-HT4 receptor (5-HT4R) agonist prucalopride can mimic this effect in mice and human. DESIGN: Using Ca2+ imaging, the effect of electrical field stimulation (EFS) and prucalopride was evaluated in situ on mMφ activation evoked by ATP in jejunal muscularis tissue. Next, preoperative and postoperative administration of prucalopride (1-5 mg/kg) was compared with that of preoperative VNS in a model of POI in wild-type and α7nAChR knockout mice. Finally, in a pilot study, patients undergoing a Whipple procedure were preoperatively treated with prucalopride (n=10), abdominal VNS (n=10) or sham/placebo (n=10) to evaluate the effect on intestinal inflammation and clinical recovery of POI. RESULTS: EFS reduced the ATP-induced Ca2+ response of mMφ, an effect that was dampened by neurotoxins tetrodotoxin and ω-conotoxin and mimicked by prucalopride. In vivo, prucalopride administered before, but not after abdominal surgery reduced intestinal inflammation and prevented POI in wild-type, but not in α7nAChR knockout mice. In humans, preoperative administration of prucalopride, but not of VNS, decreased Il6 and Il8 expression in the muscularis externa and improved clinical recovery. CONCLUSION: Enteric neurons dampen mMφ activation, an effect mimicked by prucalopride. Preoperative, but not postoperative treatment with prucalopride prevents intestinal inflammation and shortens POI in both mice and human, indicating that preoperative administration of 5-HT4R agonists should be further evaluated as a treatment of POI. TRIAL REGISTRATION NUMBER: NCT02425774.

Posterior Subdiaphragmatic Vagotomy Downmodulates the IgA Levels in the Small Intestine of BALB/c Mice.

OBJECTIVE: The posterior vagus nerve trunk innervates the entire small intestine, and elucidating its modulatory role in the IgA response was the aim of this study. METHODS: Two groups of six male BALB/c mice underwent sham or posterior subdiaphragmatic vagotomy and were euthanized on the 14th postoperative day; then, the small intestines were dissected. The intestinal fluid was harvested for antibody analysis by ELISA, and cell suspensions from Peyer's patches and lamina propria were prepared for cytofluorometric analysis of plasma cells and T lymphocytes. The CD4+ T cells were labeled for the intracellular IgA-producing interleukins (ILs)-4, -5, -6, and -10; transforming growth factor (TGF)-ß; and the inflammatory cytokines tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and IL-12. In the intestinal tissue samples, myeloperoxidase (MPO) visualization and the enzymatic activity were assessed by immunohistochemistry and ELISA, respectively. The data were analyzed by Student's t test, and the differences were considered significant at p < 0.05. RESULTS: In the vagotomy group, the IgA levels and the CD4+ T cells labeled with mediators that promote IgA secretion, including IL-4 (only at lamina propria), TNF-α, and IFN-γ, were decreased, whereas the lamina propria IgA+ plasma cells and MPO presence/activity were increased; changes in the IgM levels, IgM+ plasma cells, and CD4+ T cells labeled with TGF-ß, which have a role in class switch recombination, were not observed. CONCLUSION: The downmodulating impact of vagotomy on IgA levels may result from defective IgA secretion without affecting class switch recombination, whereas vagotomy evoked a proinflammatory response regarding MPO. These findings may reflect the role of the vagus nerve on the control of the IgA response in the small intestine.